Antibody-containing aqueous formulation and use thereof

ABSTRACT

The present disclosure provides an antibody-containing aqueous formulation, comprising a therapeutically effective amount of an anti-interleukin-6 receptor antibody, a protein stabilizer, a surfactant, and a buffer. The buffer is an acetate buffer or a histidine buffer, and the antibody-containing aqueous formulation has a pH ranging from 4.5 to 6.5.

CROSS-REFERENCE TO RELATED APPLICATION

The present disclosure claims priority to U.S. Provisional ApplicationNo. 62/651,269, filed on Apr. 2, 2018, the entirety of which isincorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to an antibody-containing aqueousformulation, and more particularly, stabilizing interleukin-6 receptorantibodies in the antibody-containing aqueous formulation for treatinginterleukin-6-mediated diseases.

BACKGROUND OF THE DISCLOSURE

Interleukin-6 (IL-6) is a cytokine that is active in inflammation. IL-6is involved in IL-6-mediated diseases, such as rheumatoid arthritis (RA)and systemic juvenile idiopathic arthritis. Hence, antibodies binding toIL-6 receptor are developed as the therapeutic agents for theIL-6-mediated diseases.

Tocilizumab IgG is a humanized monoclonal antibody against the IL-6receptor. “Actemra®” manufactured by Chugai Pharmaceutical Co., Ltd. isa FDA-approved biologic containing Tocilizumab IgG. The intravenousformulation of Actemra® comprises 20 mg/ml of Tocilizumab IgG, 5% ofsucrose, 0.05% of polysorbate 80, and 15 mM of sodium phosphate. The pHvalue of Actemra® intravenous formulation is 6.5.

However, Tocilizumab IgG formulated in Actemra® intravenous formulationis unstable when exposed to thermal stress. There is a 0.66% proteinaggregation for Tocilizumab IgG in Actemra® intravenous formulation.After being subjected to 6 months of storage at 4° C. and 25° C., thereare about 0.77% and 0.89% Tocilizumab IgG aggregations formed inActemra® formulation. The protein aggregates formed in the formulationmight induce immune response after being introduced into the human body.Therefore, there is a need to provide a more stable aqueous formulationthat stabilizes Tocilizumab IgG and suppresses the aggregate formationof Tocilizumab IgG.

BRIEF SUMMARY OF THE DISCLOSURE

An objective of the present disclosure is to reduce aggregate formationof anti-interleukin-6 receptor antibody and retain at least 90% ofmonomer form of the anti-interleukin-6 receptor antibody.

Another objective of the present disclosure is to provide a stableaqueous formulation comprising Tocilizumab IgG used for intravenousadministration.

An embodiment of the present disclosure provides an antibody-containingaqueous formulation. The antibody-containing aqueous formulationcomprises a therapeutically effective amount of an anti-interleukin-6receptor antibody, a protein stabilizer, a surfactant, and a buffer. Thebuffer is an acetate buffer or a histidine buffer, and theantibody-containing aqueous formulation falling within a pH of 4.5 to6.5.

Preferably, the anti-interleukin-6 receptor antibody is Tocilizumab IgG.

Preferably, a concentration of the Tocilizumab IgG falls within a rangeof 5 mg/ml to 30 mg/ml.

Preferably, a concentration of the Tocilizumab IgG is 20 mg/ml.

Preferably, the protein stabilizer is sucrose or trehalose, and aconcentration of the protein stabilizer falls within a range of 1% (w/v)to 10% (w/v).

Preferably, a concentration of the protein stabilizer is 7% (w/v).

Preferably, the surfactant is polysorbate 80, polysorbate 20 orpoloxamer, and a concentration of the surfactant falls within a range of0.01% (w/v) to 0.05% (w/v).

Preferably, a concentration of the surfactant is 0.03% (w/v).

Preferably, a concentration of the buffer falls within a range of 10 mMto 30 mM.

Preferably, a concentration of the buffer is 20 mM.

Preferably, the buffer is an acetate buffer, and a pH of theantibody-containing aqueous formulation falls within a range of 5.0 to6.0.

Preferably, the buffer is a histidine buffer, and a pH of theantibody-containing aqueous formulation falls within a range of 5.5 to6.5.

Preferably, the antibody-containing aqueous formulation comprises atonicifer.

Preferably, the tonicifier is 0% (w/v) to 6% (w/v) of sorbitol. Morepreferably, the tonicifier is 1% (w/v) to 4% (w/v) of sorbitol.

Preferably, the anti-interleukin-6 receptor antibody is 5 mg/ml to 30mg/ml of Tocilizumab IgG, the protein stabilizer is 4% (w/v) to 7% (w/v)of sucrose, the surfactant is 0.01% (w/v) to 0.05% (w/v) of polysorbate80, the buffer is 10 mIVI to 30 mM of acetate buffer, and theantibody-containing aqueous formulation has a pH ranging between 5.0 and6.0.

Preferably, the anti-interleukin-6 receptor antibody is 20 mg/ml ofTocilizumab IgG, the protein stabilizer is 7% (w/v) of sucrose, thesurfactant is 0.03% (w/v) of polysorbate 80, the buffer is 20 mM ofacetate buffer, and the antibody-containing aqueous formulation has a pHranging between 5.0 and 5.5

Preferably, the anti-interleukin-6 receptor antibody is 5 mg/ml to 30mg/ml of Tocilizumab IgG, the protein stabilizer is 4% (w/v) to 7% (w/v)of sucrose, the surfactant is 0.01% (w/v) to 0.05% (w/v) of polysorbate80, the buffer is 10 mM to 30 mM of histidine buffer, and theantibody-containing aqueous formulation has a pH ranging between 5.5 and6.5.

Preferably, the anti-interleukin-6 receptor antibody is 20 mg/ml ofTocilizumab IgG, the protein stabilizer is 7% (w/v) of sucrose, thesurfactant is 0.03% (w/v) of polysorbate 80, the buffer is 20 mM ofhistidine buffer, and the antibody-containing aqueous formulation has apH ranging between 5.5 and 6.5.

Preferably, the antibody-containing aqueous formulation comprises atonicifier, and the tonicifier is 0% (w/v) to 6% (w/v) of sorbitol. Morepreferably, the tonicifier is 1% (w/v) to 4% (w/v) of sorbitol.

Preferably, the antibody-containing aqueous formulation forms less than5% of aggregates of the anti-interleukin-6 receptor antibody after 3months of storage at 40° C.

Preferably, at least 90% of the anti-interleukin-6 receptor antibody inthe antibody-containing aqueous formulation is in monomer form after 3months of storage at 40° C.

Preferably, the antibody-containing aqueous formulation forms less than5% of aggregates, and at least 90% of the anti-interleukin-6 receptorantibody in the antibody-containing aqueous formulation is in monomerform after 3 months of storage at 40° C.

Another embodiment of the present disclosure provides a method oftreating an interleukin-6-mediated disease. The method comprisesadministering the antibody-containing aqueous formulation to a mammalianbody.

Another embodiment of the present disclosure provides a method ofstabilizing anti-interleukin-6 receptor antibodies. The method comprisessteps of formulating aforementioned antibody-containing aqueoussolution.

Another embodiment of the present disclosure provides a method ofsuppressing aggregate formation of the anti-interleukin-6 receptorantibodies in an antibody-containing aqueous formulation. The methodcomprises steps of formulating aforementioned antibody-containingaqueous solution.

Preferably, the antibody-containing aqueous formulation is administeredby intravenous injection or infusion.

Preferably, the interleukin-6-mediated disease is rheumatoid arthritis,systemic juvenile idiopathic arthritis, polyarticular juvenileidiopathic arthritis, Still's disease, Castleman's disease, giant cellarteritis, system sclerosis, cytokine release syndrome, polymyositis,dermatomyositis, familial mediterranean fever, polymyalgia rheumatic,Schnitzler syndrome, pulmonary arterial hypertension, pain, or B-cellchronic lymphocytic leukemia.

Another embodiment of the present disclosure provides a use of theaforementioned antibody-containing aqueous formulation in themanufacture of a medicament for treatment of an interleukin-6-mediateddisease.

Preferably, the antibody-containing aqueous formulation is administeredby intravenous injection or infusion.

Preferably, the interleukin-6-mediated disease is rheumatoid arthritis,systemic juvenile idiopathic arthritis, polyarticular juvenileidiopathic arthritis, Still's disease, Castleman's disease, giant cellarteritis, system sclerosis, cytokine release syndrome, polymyositis,dermatomyositis, familial mediterranean fever, polymyalgia rheumatic,Schnitzler syndrome, pulmonary arterial hypertension, pain, or B-cellchronic lymphocytic leukemia.

Another embodiment of the present disclosure provides a pharmaceuticalcomposition for treatment of an interleukin-6-mediated disease. Thepharmaceutical composition comprises the aforementionedantibody-containing aqueous formulation.

Preferably, the antibody-containing aqueous formulation is administeredto a mammalian body by intravenous injection or infusion.

Preferably, the interleukin-6-mediated disease is rheumatoid arthritis,systemic juvenile idiopathic arthritis, polyarticular juvenileidiopathic arthritis, Still's disease, Castleman's disease, giant cellarteritis, system sclerosis, cytokine release syndrome, polymyositis,dermatomyositis, familial mediterranean fever, polymyalgia rheumatic,Schnitzler syndrome, pulmonary arterial hypertension, pain, or B-cellchronic lymphocytic leukemia.

In sum, according to various embodiments of the present disclosure, theantibody-containing aqueous formulation having a pH ranging between 4.5and 6.5 can stabilize and reduce aggregate formation of theanti-Interleukin-6 receptor antibody in the antibody-containing aqueousformulation.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of thepresent invention and, together with the written description, explainthe principles of the present disclosure. Wherever possible, the samereference numbers are used throughout the drawings to refer to the sameor like elements of an embodiment.

FIG. 1 is a SEC-HPLC profile of Tocilizumab IgG before being subjectedto storage (To), according to an exemplary embodiment of the presentdisclosure.

FIG. 2 is a SEC-HPLC profile of Tocilizumab IgG after being subjected tostorage at 4° C. for 6 months, according to an exemplary embodiment ofthe present disclosure.

FIG. 3 is a SEC-HPLC profile of Tocilizumab IgG after being subjected tostorage at 25° C. for 6 months, according to an exemplary embodiment ofthe present disclosure.

In accordance with common practice, the various described features arenot drawn to scale and are drawn to emphasize features relevant to thepresent disclosure. Like reference characters denote like elementsthroughout the figures and text.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present disclosure will now be described more fully hereinafter withreference to the accompanying drawings illustrating various exemplaryembodiments of the present disclosure. The present disclosure may,however, be embodied in many different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the disclosure to thoseskilled in the art. Like reference numerals refer to like elementsthroughout.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the disclosure.As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” or “includes” and/or “including” or “has” and/or“having” when used herein, specify the presence of stated features,regions, integers, steps, operations, elements, and/or components, butdo not preclude the presence or addition of one or more other features,regions, integers, steps, operations, elements, components, and/orgroups thereof.

It will be understood that the terms “and/or” and “at least one” includeany and all combinations of one or more of the associated listed items.It will also be understood that, although the terms first, second, thirdetc. may be used herein to describe various elements, components,regions, parts and/or sections, these elements, components, regions,parts and/or sections should not be limited by these terms. These termsare only used to distinguish one element, component, region, part orsection from another element, component, region, layer or section. Thus,a first element, component, region, part or section discussed belowcould be termed a second element, component, region, layer or sectionwithout departing from the teachings of the present disclosure.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, such as suppression or inhibition of aninterleukin-6-mediated disease. A therapeutically effective amount of ananti-interleukin-6 receptor antibody, may vary according to factors suchas the disease state, age, gender, and weight of the subject, and theability of anti-interleukin-6 receptor antibody, to elicit a desiredresponse in the subject. Dosage regimens may be adjusted to provide theoptimum therapeutic response. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of anti-interleukin-6receptor antibody are outweighed by the therapeutically beneficialeffects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result, such as preventing an interleukin-6-mediateddisease. A prophylactically effective amount can be determined asdescribed above for the therapeutically effective amount. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount shall be lessthan the therapeutically effective amount.

It is to be noted that dosages of the anti-interleukin-6 receptorantibody may vary with the severity of the condition to be alleviated.It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the immunotherapeutic combination.

In an embodiment of the present disclosure, an antibody-containingaqueous formulation includes the therapeutically effective or theprophylactically effective amount of the anti-interleukin-6 receptorantibody, a protein stabilizer, a surfactant, and a buffer. Theantibody-containing aqueous formulation of the present disclosure has apH of 4.5 to 6.5, and the buffer is acetate buffer or histidine buffer.The antibody-containing aqueous formulation of the present disclosurecan cause less than 5% aggregate formation of the anti-interleukin-6receptor antibody after 3 months of storage at 40° C. Furthermore, theantibody-containing aqueous formulation of the present disclosure canretain at least 90% of monomer form of the anti-interleukin-6 receptorantibody after 3 months of storage at 40° C. The present disclosure willbe further illustrated below with reference to specific examples, andwithin each of the examples, several formulation candidates withdifferent compositions or pH values are provided. The following examplesand materials described hereafter are for exemplary purposes only.

1. Material and Methods

In following formulation candidates provided by the present disclosure,Tocilizumab IgG is expressed in CHO-DG44 cells. CHO-DG44 cells arecultured in a 2,000-liter bioreactor, and fed-batch process wasconducted. Tocilizumab IgG generated by the above process is purified bya standard series of chromatography steps known in the art, includingrPA affinity chromatography, ion-exchange chromatography, and mix-modechromatography. Tangential Flow Filtration is further performed toconcentrate the above purified IgG proteins using ultra-filtrationmembrane, and a diafiltration is conducted to exchange selected buffer.

To assess buffer system affecting the stability and aggregate formationof Tocilizumab IgG, 20 mg/ml Tocilizumab IgG is formulated in variousbuffers (e.g., acetate buffer or histidine buffer) with addition ofexcipients. The commercially available Acterma® is used as a controlgroup in the present disclosure. Acterma® comprises 20 mg/ml ofTocilizumab IgG, 15 mM of sodium phosphate buffer at pH 6.5, 5% ofsucrose, and 0.05% of polysorbate 80.

The excipients are the protein stabilizer and the surfactant. Theprotein stabilizer may be sucrose or trehalose. The surfactant is notlimited to polysorbate 80, polysorbate 20, or poloxamer.

Thermal stress, mechanical stress, oxidation stress, photo stress, andfreeze-thaw stress are performed to evaluate the stability ofTocilizumab IgG in the formulation candidates. The conditions of thesestress tests are listed in Table 1.

TABLE 1 The conditions of the thermal, mechanical, oxidative, photo andfreeze- thaw stress tests. Time Stress Tests Condition Point(s) Thermalstress 4° C. or 40° C. 0, 8 weeks, 3 months or 6 months Mechanicalstress Agitation with mini vortexer 4 hr constant agitation Oxidationstress Tertiary Butyl Peroxide added at 24 hrs 1% w/v Photo stressUV-light exposure (broad spectrum) 24 hrs Freeze-thaw −70° C. to RoomTemperature 5 consecutive stress cycles

1.1. IL-6R Binding Assay (Potency)

After purification or stress treatment, the samples containingTocilizumab IgG are filled into the 96-well plate, of which the surfaceis coated with IL-6 receptors. After incubating for 24 hours, thebinding reaction is terminated by adding acid solution, and bindingefficiency is further analyzed.

1.2. Size Exclusion Chromatography-High Performance LiquidChromatography (SEC-HPLC)

SEC-HPLC is used to monitor protein aggregation and fragmentation of theformulation candidates of the present disclosure, and determine thepurity of Tocilizumab IgG of the formulation candidates of the presentdisclosure. About 200 μg of Tocilizumab IgG of each sample after stresstreatment is injected for SEC-HPLC analysis.

FIGS. 1-3 are SEC-HPLC profiles, in accordance with an embodiment of thepresent disclosure. In FIGS. 1-3, main peaks represent monomers of theTocilizumab IgG; pre-peaks represent high molecular weight (HMW)aggregate formation of Tocilizumab IgG. Aggregate formation ofTocilizumab IgG increases risks for inducing immune response after beingintroduced into the human body. In FIGS. 1-3, post-peaks representdegraded low molecular weight (LMW) fragments of Tocilizumab IgG. Thehigher the main peaks represents higher purity and stability of theformulation.

1.3. FlowCam Analysis

The FlowCam analysis is used to monitor the sub-visible particles formedin the formulation comprising Tocilizumab IgG. According to USParmacopeia Chapter <787> (USP<787>), the count of sub-visible particlesof the therapeutic protein injection should be analyzed, and particlesfell into the size range of >10 μm and >25 μm would be defined assub-visible particles. Based on USP<787>, the count of particles withthe size larger than 10 μm must be less than 6,000 particles/container,and the count of particles with the size larger than 25 μm must be lessthan 600 particles/container if the container volume is smaller than 100mL. Larger sub-visible particles represent higher risk ofimmunogenicity. The FlowCam analysis is performed with the parametersdetailed in Table 3.

TABLE 3 The conditions for FlowCam analysis Particle Segmentation DarkThreshold 15.00; Light Threshold 15.00 Distance to Nearest Neighbor 0microns Close Holes 5 iterations Basic Size Filter equivalent sphericaldiameter (ESD): Min 1.00, Max 10000.00 microns Advanced Filter NoneAuto-Image Frame Rate 39 frames per second Flash Duration 300.00microseconds Camera Gain 0

2. Example 1: Effect of the Buffer System and Tonicifier on theStability of Tocilizumab IgG

To assess the impact of buffer system and tonicifier on the proteinstability of Tocilizumab IgG, 20 mg/ml Tocilizumab IgG is formulated invarious buffers (20 mM acetate, pH 4.5 or pH 5; 20 mM histidine, pH 6)with the addition of excipients containing 7% sucrose and 0.003%polysorbate 80. The buffers further include a tonicifier selected from110 mM NaCl or 4% sorbitol. These prepared formulation candidates listedin Table 2 are subjected to thermal stress, mechanical stress, oxidativestress, photo stress and freeze-thaw stress tests to evaluate thestability of Tocilizumab IgG. The commercially available Actemra®, whichcomprises 20 mg/ml of Tocilizumab IgG, 15 mM of sodium phosphate bufferat pH 6.5, 5% of sucrose, and 0.05% of polysorbate 80, is taken as theReference Medicinal Product (RMP).

TABLE 4 The composition of formulation candidates of Example 1comprising Tocilizumab IgG. Sample Target Tonicity PolysorbateFormulation Description Buffer pH modifier Stabilizer(s) 80 (w/v %) 1A4.5N 20 mM Na 4.5 110 mM 7% Sucrose 0.03 Acetate NaCl 2 A4.5S 20 mM Na4.5 4% 7% Sucrose 0.03 Acetate Sorbitol 3 A5.0N 20 mM Na 5 110 mM 7%Sucrose 0.03 Acetate NaCl 4 A5.0S 20 mM Na 5 4% 7% Sucrose 0.03 AcetateSorbitol 5 H6N 20 mM 6 110 mM 7% Sucrose 0.03 Histidine NaCl 6 H6S 20 mM6 4% 7% Sucrose 0.03 Histidine Sorbitol 7 Actemra ® 15 mM Na 6.5 5%Sucrose 0.05% PS DP Phosphate 80

2.1. Thermal Stress Test

The prepared formulation candidates are subjected to a storage time of 8weeks (or 2 months) at −70° C., 4° C. or 40° C., and followed by furtheranalysis. The following analyses after storage are pH value assay,amniotic fluid optical density at 650 nm (OD₆₅₀), protein concentrationassay, binding ability assay, SEC-HPLC analysis, and FlowCam analysis.

2.1.1. pH, OD₆₅₀ and Protein Concentration Assay After Thermal StressTest

The formulation candidates are analyzed for the pH value, proteinconcentration and amniotic fluid optical density at 650 nm (OD₆₅₀)following the 8 weeks of storage at the either 4° C. or 40° C. As shownin Table 5, the pH value, OD₆₅₀ value and protein concentration of eachformulation candidate of the present disclosure has no significantchange after being treated with thermal stress.

TABLE 5 pH, OD₆₅₀ and protein concentration of the formulationcandidates of Example 1 Item pH value OD₆₅₀ Concentration (mg/mL)Temperature T₀ 8 weeks T₀ 8 weeks T₀ 8 weeks (control) 4° C. 40° C.(control) 4° C. 40° C. (control) 4° C. 40° C. A4.5N 4.52 4.46 4.60 0.0060.004 0.005 20.57 20.77 20.77 A4.5S 4.61 4.47 4.66 0.002 0.001 0.01220.62 20.89 20.82 A5.0N 4.96 4.92 4.99 0.002 0.003 0.003 20.64 19.9120.72 A5.0S 5.02 4.94 5.05 0.000 −0.010 −0.009 20.36 20.41 20.72 H6N6.16 6.03 6.07 0.000 0.009 0.001 21.67 21.88 19.53 H6S 6.12 6.10 6.080.003 0.002 0.002 19.21 19.39 19.53 Actemra ® 6.52 6.50 6.51 0.000 0.0010.002 19.38 19.48 19.682.1.2. Potency of Tocilizumab IgG after Thermal Stress Test

The relative binding capacity of Tocilizumab IgG formulated in theformulation candidates of the present disclosure after being treatedwith different temperatures of thermal stress was measured by usingenzyme-linked immunosorbent assay (ELISA).

As shown in Table 6, the binding activity of Tocilizumab IgG formulatedin different buffer (including Actemra®) has no significant differenceafter being subjected to 8 weeks of storage at −70° C. or 4° C. comparedwith samples before being subjected to storage, but it slightlydecreased after the storage at 40° C.

Further, Tocilizumab IgG formulated in A4.5N, A4.5S, A5.0N, A5.0S, H6Nand H6S shows higher binding activity value than that formulated in theRMP, Actemra®, after being treated with thermal stress at 40° C.,indicating that Tocilizumab IgG formulated in the above describedformulation presented better thermostability than that formulated inActermra®.

TABLE 6 Potency of Tocilizumab IgG in the formulation candidates ofExample 1 after thermal stress Potency % Temperature Item T₀ −70° C. 4°C. 25° C. 40° C. A4.5N 102.5 116.3 109.3 112.6 98.8 A4.5S 119.8 105111.5 114.9 100.1 A5.0N 122.6 103.4 113.9 105.7 98 A5.0S 117.6 96.6104.5 108.7 102.3 H6N 102.3 105.8 98.7 96 98 H6S 95.2 110.9 104.7 108.895.8 Actemra ® 115.7 114.4 123 119 94.22.1.3. SEC-HPLC Analysis after Thermal Stress Test

Tables 7 to 9 demonstrate the percentages of Tocilizumab IgG monomers(main peak %), aggregates (pre-peak %), and fragments (post-peak %)determined by the SEC-HPLC before being subjected to storage (To) orafter being subjected to 8 weeks of storage at either 4° C. or 40° C.

In addition, the results in Table 9 indicate that formulations of H6N(95.34%) and H6S (95.18%) show higher percentage of main peak thanActemra® (94.67%) after storage for 8 weeks at 40° C.

Tables 7 to 9 also show that formulations comprising sorbitol has bothless HMW aggregates (HMW %) and less aggregation increase (increased HMW%) than those containing 110 mM sodium chloride after thermal stresstreatment. Further, the A4.5N formulation shows high decrease in mainpeak % and high increase in HMW % and LMW % after storage at 40° C.Based on the data, it is found that the presence of sorbitol improvedthe stability of Tocilizumab IgG, especially in low pH environment.However, a new generated pre-peak 3 (HMW aggregates) is detected inA4.5N and A4.5S formulations after a storage condition of 40° C. for 8weeks.

In general, comparing to Actemra® (RMP), Tocilizumab IgG has lessaggregation and similar or higher monomer-form when formulated inacetate buffer at pH 5 or in histidine buffer at pH 6 containing 4%sorbitol. Even under the storage condition of 40° C. for 8 weeks, theformulation of acetate buffer at pH 5 or histidine buffer at pH 6comprising 4% sorbitol retains >90% monomers and forms <1% aggregates.The result indicates that Tocilizumab IgG formulated in the buffer ofacetate (pH 5) or histidine (pH 6) comprising 4% of sorbitol, 7% ofsucrose and 0.003% of polysorbate 80 has better thermostability thanTocilizumab IgG formulated in Actemra®.

TABLE 7 SEC-HPLC peak summary of the formulation candidates of Example 1Pre-Peak 1% Main Peak % Post peak % Sum area T₀-A4.5N 0.19 99.49 0.3340048 T₀-A4.5S 0.14 99.46 0.41 41047 T₀-A5.0N 0.23 99.49 0.29 43137T₀-A5.0S 0.15 99.43 0.42 41540 T₀-H6N 0.29 99.45 0.27 44002 T₀-H6S 0.2599.45 0.31 38899 T₀-RMP 0.88 98.84 0.28 42739

TABLE 8 SEC-HPLC peak summary of the formulation candidates of Example1, with the storage condition of 4° C. for 8 weeks Increased Post- Pre-Increased Main Main- Increased peak Post- Post- Sum peak1 % HMW % HMW %peak % peak % LMW % LMW % 1 % peak1′ peak area 4C8W-A4.5N 0.24 0.24 0.0499.53 −0.13 0.23 0.09 0.23 0.05 0.18 38682 4C8W-A4.5S 0.15 0.15 −0.0199.69 0.00 0.16 0.02 0.16 — 0.16 38159 4C8W-A5.0N 0.31 0.31 0.06 99.49−0.12 0.20 0.06 0.20 0.05 0.15 37565 4C8W-A5.0S 0.18 0.18 0.00 99.62−0.05 0.20 0.06 0.20 0.05 0.15 37936 4C8W-H6.0N 0.34 0.34 0.01 99.47−0.06 0.20 0.06 0.20 0.06 0.14 40959 4C8W-H6.0S 0.28 0.28 −0.02 99.51−0.05 0.21 0.07 0.21 0.06 0.15 35637 4C8W-ACTEMRA ® 0.72 0.72 0.02 99.16−0.03 0.12 0.01 0.12 — 0.12 36038

TABLE 9 SEC peak summary of the formulation candidates of Example 1,with the storage condition of 40° C. for 8 weeks Pre- Pre- Pre- MainIncreased Shoulder Post- Post- peak3 peak2 peak1 HMW Increased peakMain- LMW Increased peak peak Post- Post- peak2 Sum % % % % HMW % % peak% % LMW % % 1 % peak1 peak1′ % area 40C8W-A4.5N 0.04 — 1.19 1.25 1.0338.35 −11.31 10.42  10.28  7.19 2.90 0.13 2.77 0.33 37940 40C8W-A4.5S0.03 — 0.29 0.26 0.10 93.22 −6.47 6.53 6.39 4.82 1.71 0.09 1.62 — 3785340C8W-A5.0N — — 0.80 0.80 0.55 92.93 −8.68 6.27 6.13 4.65 1.62 0.08 1.54— 38164 40C8W-A5.0S — — 0.31 0.31 0.33 94.66 −5.01 5.03 4.39 3.80 1.230.06 1.17 — 37948 40C8W-H6.0N — — 0.56 0.56 0.25 96.34 −4.19 4.09 3.953.17 0.92 0.05 0.87 — 40112 40C8W-H6.0S — — 0.40 0.40 0.10 95.18 −4.384.43 4.27 3.46 0.95 0.05 0.90 — 35779 40C8W- — — 1.03 1.03 0.33 94.67−4.52 4.29 4.18 3.35 0.94 — 0.94 — 35886 ACTEMRA ®2.1.4. FlowCam Analysis after Thermal Stress Test

Tables 10 to 12 demonstrate the particle counts (the quantity ofparticles per milliliter, P/ML) of the formulation candidates determinedby FlowCam analysis before being subjected to storage (To) or afterbeing subjected to 8 weeks of storage at either 4° C. or 40° C. Thenumbers described in Tables represent the quantity of particles permilliliter in each formulation treated with thermal stress at selectedtemperature.

In Table 10 and Table 12, the formulations of A4.5S, A5.0S, H6N and H6Sshow similar particle count to Actemra®, before being subject tostorage. After 8 weeks of storage at either 4° C., the formulations ofA4.5N, A4.5S, A5.0N, A5.0S, and H6S are determined lower particle countin the size ranges of >10 μm and >25 μm than Actemra®. After 8 weeks ofstorage at 40° C., the formulations of A4.5N, A4.5S, A5.0N, A5.0S, H6Nand H6S are determined lower particle count in the size ranges of >10 μmand >25 μm than Actemra®.

Based on Tables 10 to 12, the formulation candidates A4.5S, A5.0S, H6Nand H6S show similar or even lower particle count in the size rangesof >10 μm and >25 μm to the Actemra® (RMP) before being subjected tostorage (To) or after being subjected to 8 weeks of storage at either 4°C. or 40° C., indicating that these formulation might improve thethermostability of Tocilizumab IgG.

TABLE 10 FlowCam results of the formulation candidates of Example 1after thermal stress test, before subjected to storage (T₀) P/ML A4.5NA4.5S A5.0N A5.0S H6N H6S RMP 1-2 μm 6237 2379 3418 3235 6198 4489 71252-4 μm 6394 2041 4516 2838 5206 4567 6078 4-6 μm 862 393 763 549 828 7221205 6-8 μm 501 212 519 244 205 306 520 8-10 μm 204 71 244 61 90 102 24410-25 μm 368 141 549 122 98 118 87 >25 μm 63 71 31 61 25 55 16

TABLE 11 FlowCam results of the formulation candidates of Example 1after thermal stress, with a storage condition of 4° C. for 8 weeks 4C8WA4.5N A4.5S A5.0N A5.0S H6N H6S RMP 1-2 μm 3198 1420 3816 2303 2227 23786309 2-4 μm 2452 1095 3465 1356 2336 2154 6578 4-6 μm 347 145 538 165447 355 822 6-8 μm 160 95 255 110 243 126 389 8-10 μm 24 32 67 28 122 91185 10-25 μm 45 138 63 48 263 189 193 >25 μm 12 43 19 0 118 44 8

TABLE 12 FlowCam results of the formulation candidates of Example 1after thermal stress, with a storage condition of 40° C. for 8 weeks40C8W A4.5N A4.5S A5.0N A5.0S H6N H6S RMP 1-2 μm 2973 1575 1471 20845770 5076 8758 2-4 μm 3417 1051 1318 1435 5946 5032 9464 4-6 μm 683 158212 94 1215 834 1807 6-8 μm 271 47 75 63 463 331 740 8-10 μm 142 12 8 20169 90 292 10-25 μm 154 31 16 43 192 67 347 >25 μm 4 12 8 19 31 12 59

2.2. Freeze-Thaw Stress Test

The prepared formulation candidates are performed with freeze-thawprocess. The process is accomplished by freezing the formulation at −70°C. and subsequently thawing the ice to room temperature. The cycle offreezing and thawing is repeated for 5 times consecutively. Then, theprocess followed by further analyses, including pH value assay, amnioticfluid optical density at 650 nm (OD₆₅₀), protein concentration assay,SEC-HPLC analysis, and FlowCam analysis.

2.2.1. pH, OD₆₅₀ and Protein Concentration Assay After Freeze/ThawStress Test

According to data shown in Table 13, the pH value, OD₆₅₀ value andprotein concentration of each of the formulation candidates has nosignificant change after freeze-thaw tests.

TABLE 13 pH, OD₆₅₀ and protein concentration assay results of theformulation candidates of Example 1, after freeze-thaw stress tests pHvalue OD₆₅₀ Concentration (mg/mL) Temperature control Experiment controlExperiment control Experiment A4.5N 4.51 4.56 0.001 0.003 20.76 20.675A4.5S 4.61 4.64 0.007 0.000 20.76 20.692 A5.0N 5.00 4.95 0.003 0.00320.74 20.708 A5.0S 5.04 5.01 0.000 0.000 20.76 20.639 H6N 6.09 6.100.003 0.001 21.83 21.621 H6S 6.11 6.13 0.000 0.000 19.46 19.234Actemra ® 6.54 6.51 0.002 0.002 19.34 19.4692.2.2. SEC-HPLC Analysis after Freeze-Thaw Stress Test

Table 14 shows SEC-HPLC measurements of the purity of Tocilizumab IgGformulated in the formulation candidates of the present disclosure.These measurements include the amount of monomer (main peak %) ofremaining, the amount of aggregates (HMW %) and fragments (LMW %) formedin the samples and the increase in aggregates (increased HMW %) andfragments (increased LMW %).

The results described in Table 14 show that there were no obviousaggregates increase (increased HMW %) and fragments increase (increasedLMW %) of Tocilizumab IgG in the tested formulation candidates afterfreeze-thaw tests.

TABLE 14 SEC-HPLC peak summary of the formulation candidates of Example1, after freeze-thaw stress test Pre- Increased Post- peak IncreasedMain Main- Increased peak 1 % HMW % HMW % peak % peak % LMW % LMW % 1 %Sum area FT5-A4.5N 0.2 0.20 0.00 99.70 0.04 0.10 −0.04 0.10 38800340FT5-A4.5S 0.15 0.15 −0.01 99.75 0.06 0.09 −0.05 0.09 37249128 FT5-A5.0N0.24 0.24 −0.01 99.66 0.05 0.09 −0.05 0.09 38095255 FT5-A5.0S 0.17 0.17−0.01 99.73 0.06 0.09 −0.05 0.09 37348239 FT5-H6.0N 0.33 0.33 0.00 99.580.05 0.09 −0.05 0.09 39220858 FT5-H6.0S 0.28 0.28 −0.02 99.63 0.07 0.10−0.04 0.10 34769108 FT5- 0.66 0.66 −0.04 99.26 0.07 0.08 −0.03 0.0835072563 ACTEMRA ®2.2.3. FlowCam Analysis after Freeze-Thaw Stress Test

The formulation candidates of the present disclosure treated withfreeze-thaw cycles is further evaluated by using FlowCam analysis, toacquire particle count data. The particle count of the formulationcandidates before being subjected to freeze-thaw processing (Control) isshown in Table 10. Tables 15 provides the particle count of theformulation candidates after being subjected to freeze/thaw cycles, andthe formulations of A4.5N, A4.5S, A5.0N, A5.0S, and H6N are determinedlower particle count in the size ranges of >10 μm and >25 μm thanActemra® (RMP).

Based on the data presented in Tables 10 and 15, it is found that theformulations candidates A5.0S and H6N show similar or even lowerparticle count in the size ranges of >10 μm and >25 μm to Actemra® (RMP)before or after being subjected to freeze-thaw cycles, indicating thatthese formulation candidates might prevent sub-visible particleformation of Tocilizumab IgG.

TABLE 15 FlowCam results of the formulation candidates of Example 1,after freeze-thaw tests P/ML A4.5N A4.5S A5.0N A5.0S H6N H6S RMP 1-2 μm4177 1725 1901 3791 1356 4819 15478 2-4 μm 4993 2000 2051 5375 1790 501315425 4-6 μm 1205 617 413 1674 315 765 2187 6-8 μm 644 327 248 601 127183 1026 8-10 μm 412 141 98 128 135 91 389 10-25 μm 269 268 368 158 427365 704 >25 μm 0 30 0 0 0 206 0

2.3. Mechanical Stress Test: Agitation

Agitation is a form of mechanical stress, and can lead to theaggregation of the protein molecules. The formulation candidates areperformed with agitation and followed by further analysis, including pHvalue assay, amniotic fluid optical density at 650 nm (OD₆₅₀), proteinconcentration assay, SEC analysis, and FlowCam analysis.

2.3.1. pH, OD₆₅₀ and Protein Concentration Assay after Mechanical StressTest

As shown in Table 16, the pH value, OD₆₅₀ value and proteinconcentration of each of the formulation candidates has no significantchanges after the treatment of agitation.

TABLE 16 pH, OD₆₅₀ and protein concentration assay results of theformulation candidates of Example 1, after mechanical stress test(agitation) pH value OD₆₅₀ Concentration (mg/mL) Temperature controlExperiment control Experiment control Experiment A4.5N 4.51 4.5 0.0010.002 20.76 20.63 A4.5S 4.61 4.61 0.007 0.009 20.76 20.71 A5.0N 5.004.98 0.003 0.004 20.74 20.71 A5.0S 5.04 5.03 0.000 0.000 20.76 20.55 H6N6.09 6.06 0.003 0.001 21.83 21.65 H6S 6.11 6.10 0.000 0.000 19.46 19.19Actemra ® 6.54 6.51 0.002 0.002 19.34 19.432.3.2. SEC Analysis after Mechanical Stress Test

Table 17 demonstrates the SEC-HPLC analyses results of the formulationcandidates, after being subjected to agitation. The results of SEC-HPLCanalyses show that there was no significant change in the amount ofmonomer (main peak %), aggregates (HMW %) and fragments (LMW %),indicating that there were no aggregate increase (increased HMW %) andfragment increase (increased LMW %) of Tocilizumab Ig after agitationstress.

TABLE 17 SEC-HPLC analyses results of the formulation candidates ofExample 1, after mechanical stress test (agitation) Increased Pre-Increased Main Main- Increased Post- peak1 % HMW % HMW % peak % peak %LMW % LMW % peak % Sum area AGIT- 0.22 0.22 0.01 99.65 −0.04 0.13 0.030.13 37655343 A4.5N AGIT- 0.14 0.14 0.00 99.73 −0.02 0.13 0.02 0.1336906315 A4.5S AGIT- 0.27 0.27 0.02 99.61 −0.04 0.11 0.02 0.11 37506113A5.0N AGIT- 0.15 0.15 −0.01 99.68 −0.06 0.16 0.06 0.16 38434929 A5.0SAGIT- 0.30 0.30 0.01 99.59 −0.03 0.11 0.02 0.11 40258821 H6.0N AGIT-0.26 0.26 0.01 99.62 −0.03 0.13 0.02 0.13 35762186 H6.0S AGIT- 0.65 0.650.00 99.27 −0.01 0.08 0.01 0.08 36717662 Actemra ®

2.3.3. FlowCam Analysis After Mechanical Stress Test (Agitation)

Tables 18 and 19 demonstrate the particle counts of the formulationcandidates by using FlowCam analysis before or after being subjected toagitation. The formulation candidates that are not subjected toagitation are labeled as non-agitation group in Table 18. Tables 18 and19 show that the formulations candidates A4.5N, A4.5S, A5.0N, A5.0S andH6S in groups with agitation and without agitation presented similar oreven lower number of particles per milliliter in the size ranges of >10μm and >25 μm, when comparing with Actemra® (RMP). Further, there was noincrease in the particle count in the size ranges of interest (>10 μmand >25 μm) in the FlowCam results of the formulation candidates A4.5N,A5.0S and H6S, when comparing to the non-agitation group.

TABLE 18 FlowCam results of the formulation candidates of Example 1without agitation control A4.5N A4.5S A5.0N A5.0S H6N H6S RMP 1-2 μm3126 1972 2193 3745 5186 2074 4881 2-4 μm 2704 3010 1960 4036 5193 41864836 4-6 μm 435 621 270 646 1316 2486 931 6-8 μm 198 322 113 277 11581221 387 8-10 μm 106 31 45 66 906 374 156 10-25 μm 119 31 38 158 1025180 134 >25 μm 0 0 0 0 16 0 0

TABLE 19 FlowCam results of the formulation candidates of Example 1 withagitation Agitation A4.5N A4.5S A5.0N A5.0S H6N H6S RMP 1-2 μm 5114 615515903 4652 5733 5289 10581 2-4 μm 2978 3859 14683 2974 8679 5170 104174-6 μm 238 486 2492 360 951 767 1841 6-8 μm 52 172 723 142 225 253 7918-10 μm 15 82 211 105 106 82 274 10-25 μm 22 45 151 90 145 164 125 >25μm 0 0 0 0 0 0 8

2.4. Photo Stress Test

The formulation candidates are exposed to UV light for 5 hours with thepower of 1.6 watts/hour and followed by further analyses, and theseanalyses includes pH value assay, amniotic fluid optical density at 650nm (OD₆₅₀), protein concentration assay, and SEC-HPLC analysis.

2.4.1: pH, OD₆₅₀ and Protein Concentration Assay after Photo Stress Test

According Table 20, the pH value, OD₆₅₀ value and protein concentrationof each of the formulation candidates has no significant change afterphoto stress test.

TABLE 20 pH, OD₆₅₀ and protein concentration of the formulationcandidates of Example 1, after photo stress test pH value OD₆₅₀Concentration (mg/mL) Temperature control Experiment control Experimentcontrol Experiment A4.5N 4.50 4.52 0.001 0.000 20.76 20.63 A4.5S 4.614.60 0.000 0.001 20.76 20.71 A5.0N 4.98 5.00 0.000 0.000 20.74 20.71A5.0S 5.03 5.03 0.004 0.003 20.76 20.55 H6N 6.09 6.08 0.003 0.004 21.8321.65 H6S 6.14 6.13 0.001 0.003 19.46 19.19 Actemra ® 6.52 6.53 0.0050.007 19.34 19.432.4.2. SEC Analysis after Photo Stress Test

Table 21 demonstrates SEC-HPLC peak summary of formulation candidatesafter being subjected to the photo stress test. The results described inTable 21 indicate that there was no significant change in the amount offragments (i.e., no significant increase of fragments (increased LMW %))of Tocilizumab IgG in each formulations after UV light exposure, andwere also no significant change in the amount of protein monomers (Mainpeak %) and aggregates (HMW %) in the formulations, including A4.5N,A4.5S, A5.0N, A5.0S, H6N and H6S. The formulations of RMP (Actemra®,pH6.5) showed slight increase (about 0.5%) in aggregates level (HMW %)and slight decrease in monomers level (Main peak %) after UV lightexposure.

TABLE 21 SEC-HPLC peak summary of the formulation candidates of Example1, after photo stress test Pre- Increased peak Increased Main Main-Increased Post- 1 % HMW % HMW % peak % peak % LMW % LMW % peak % Sumarea PHOTO- 0.24 0.24 0.02 99.63 −0.02 0.13 0.00 0.13 37017495 A4.5NPHOTO- 0.15 0.15 0.00 99.70 −0.02 0.15 0.02 0.15 36570334 A4.5S PHOTO-0.31 0.31 0.05 99.57 −0.05 0.12 0.00 0.12 38692464 A5.0N PHOTO- 0.170.17 0.01 99.69 −0.02 0.14 0.01 0.14 36401540 A5.0S PHOTO- 0.35 0.350.04 99.52 −0.05 0.13 0.01 0.13 38673652 H6.0N PHOTO- 0.29 0.29 0.0399.58 −0.02 0.13 0.00 0.13 33919777 H6.0S PHOTO- 1.20 1.20 0.52 98.71−0.53 0.09 0.01 0.09 34732505 ACTEMRA ®

2.5. Oxidation Stress Test

The formulation candidates of the present disclosure are exposed to 1%tert-butyl hydroperoxide (tBHP, the oxidant) for 24 hours and followedby further analyses. These analyses include pH value assay, amnioticfluid optical density at 650 nm (OD650), protein concentration assay,and SEC-HPLC analysis. Another group of the formulation candidates arenot exposed to tBHP, and are designated as non-oxidation group.

2.5.1. pH, OD650 and Protein Concentration Assay after Oxidation StressTest

According to Table 22, there are no significant differences among the pHvalue, OD₆₅₀ value and protein concentration of each of the formulationcandidates after exposure to the oxidant, when comparing withnon-oxidation group.

TABLE 22 pH, OD₆₅₀ and protein concentrations of the formulationcandidates of Example 1, after oxidation stress test pH value OD₆₅₀Concentration(mg/mL) non- non- non- Item oxidation Experiment oxidationExperiment oxidation Experiment A4.5N 4.51 4.52 0.003 0.004 20.39 19.56A4.5S 4.61 4.61 0.002 0.003 20.36 19.68 A5.0N 5.09 5.01 0.004 0.00419.68 19.75 A5.0S 5.05 5.05 0.002 0.002 20.10 19.53 H6N 6.05 6.11 0.0030.002 20.85 20.74 H6S 6.09 6.17 0.005 0.001 18.65 18.31 Actemra ® 6.496.51 0.006 0.004 19.12 18.452.5.2. SEC Analysis after Oxidation Stress Test

Table 23 demonstrates the results of the SEC-HPLC analyses of theformulation candidates after exposure to the oxidant. The resultsdescribed in Table 23 show that the tested formulations have nosignificant change in the amount of monomers (main peak %), aggregates(HMW %) and fragments (LMW %) (i.e., no significant increase or decreaseof monomers, aggregates and fragments) of Tocilizumab IgG after exposureto the oxidant.

TABLE 23 SEC-HPLC peak summary of the formulation candidates of Example1, after oxidation stress test Pre- Increased peak Increased Main Main-Increased Post- 1 % HMW % HMW % peak % peak % LMW % LMW % peak % Sumarea OXI-A4.5N 0.26 0.26 0.05 99.61 −0.05 0.13 0.00 0.13 36856078OXI-A4.5S 0.14 0.14 0.00 99.74 0.00 0.11 −0.01 0.11 38535448 OXI-A5.0N0.28 0.28 0.02 99.61 −0.01 0.11 0.00 0.11 37557108 OXI-A5.0S 0.17 0.170.01 99.73 0.00 0.11 0.00 0.11 37303390 OXI-H6.0N 0.31 0.31 −0.02 99.590.02 0.11 0.00 0.11 39217816 OXI-H6.0S 0.25 0.25 −0.01 99.64 0.00 0.110.00 0.11 36039515 OXI- 0.65 0.65 −0.01 99.27 0.02 0.08 −0.01 0.0834532137 ACTEMRA ®

Based on the data obtained from various stress tests, Tocilizumab IgGformulated in acetate (pH 5.0) or histidine (pH 6.0) buffer has loweraggregate formation and sub-visible particles than those formulated inActemra® (phosphate buffer at pH 6.5), after being subjected to along-term storage at 40° C., a freeze-thaw processing, agitation stress,photo stress test, and oxidation stress test. Furthermore, after beingsubjected to a long-term storage condition of 40° C. and a freeze-thawprocessing, the formulation candidates comprising 4% sorbitol havehigher remaining Tocilizumab IgG monomers and less aggregate formation,than the formulations comprising 110 mM of sodium chloride.

As evidenced by the above data presented in Example 1, acetate orhistidine buffer at pH5.0 to pH6.0 can be appropriate buffers toformulate Tocilizumab IgG. These buffers improve the stability of theantibody, and the addition of sorbitol might reduce the proteinaggregation.

3. Example 2: Effect of pH and Sorbitol Concentration on the Stabilityof Tocilizumab IgG

In Example 2 of the present disclosure, acetate is chosen as the buffersolution and further elucidate the effect of pH levels and sorbitolconcentrations on the stability of Tocilizumab IgG. Table 24 lists theformulation candidates of Example 2 (hereinafter “acetate samples”). Theformulation candidates of Example 2 comprise 20 mg/ml of Tocilizumab IgGwith varying pH (ranging from pH 5.1 to 5.4) and sorbitol concentration(ranging from 1% to 4%). Once formulated, these acetate samples arestored at 4° C. for 3 to 6 months, at 25° C. for 3 to 6 months, or at40° C. for 3 months (thermal stress test), and are subject to furtheranalyses.

TABLE 24 The compositions of the formulation candidates of Example 2Target protein Sample Target Sorbitol Polysorbate conc. Formulation #Description Buffer pH (w/v %) Stabilizer 80 (w/v %) (mg/mL) 1 A5.1S1 20mM 5.1 1% 7% 0.03 20 Na Sorbitol Sucrose Acetate 2 A5.1S2 20 mM 5.1 2%7% 0.03 20 Na Sorbitol Sucrose Acetate 3 A5.1S3 20 mM 5.1 3% 7% 0.03 20Na Sorbitol Sucrose Acetate 4 A5.1S4 20 mM 5.1 4% 7% 0.03 20 Na SorbitolSucrose Acetate 5 A5.2S1 20 mM 5.2 1% 7% 0.03 20 Na Sorbitol SucroseAcetate 6 A5.2S2 20 mM 5.2 2% 7% 0.03 20 Na Sorbitol Sucrose Acetate 7A5.2S3 20 mM 5.2 3% 7% 0.03 20 Na Sorbitol Sucrose Acetate 8 A5.2S4 20mM 5.2 4% 7% 0.03 20 Na Sorbitol Sucrose Acetate 9 A5.3S1 20 mM 5.3 1%7% 0.03 20 Na Sorbitol Sucrose Acetate 10 A5.3S2 20 mM 5.3 2% 7% 0.03 20Na Sorbitol Sucrose Acetate 11 A5.3S3 20 mM 5.3 3% 7% 0.03 20 NaSorbitol Sucrose Acetate 12 A5.3S4 20 mM 5.3 4% 7% 0.03 20 Na SorbitolSucrose Acetate 13 A5.4S1 20 mM 5.4 1% 7% 0.03 20 Na Sorbitol SucroseAcetate 14 A5.4S2 20 mM 5.4 2% 7% 0.03 20 Na Sorbitol Sucrose Acetate 15A5.4S3 20 mM 5.4 3% 7% 0.03 20 Na Sorbitol Sucrose Acetate 16 A5.4S4 20mM 5.4 4% 7% 0.03 20 Na Sorbitol Sucrose Acetate3.1 pH, OD₆₅₀ and Protein Concentration assay after Thermal Stress Test

The acetate samples are analyzed for the pH value, proteinconcentration, and amniotic fluid optical density at 650 nm (OD₆₅₀)following the 3 to 6 months of storage at the 4° C., 25° C., or 40° C.The storage conditions with different temperatures are thermal stresstests for the acetate samples.

As shown in Tables 25, after a long-term storage at a lower temperature(4° C.), room temperature (25° C.) or elevated temperature (40° C.), thepH value, OD₆₅₀ value and protein concentration of each of theformulation candidates has no significant change.

TABLE 25 pH, OD₆₅₀ and protein concentrations assay of the formulationcandidates of Example 2, after thermal stress test pH valueConcentration (mg/mL) Item T₀ 3 months 6 months T₀ 3 months 6 monthsTemperature (control) 4° C. 25° C. 40° C. 4° C. 25° C. (control) 4° C.25° C. 40° C. 4° C. 25° C. A5.1S1 5.08 5.14 5.14 5.14 5.12 5.13 20.10819.972 20.004 19.401 19.851 19.917 A5.1S2 5.11 5.13 5.13 5.12 5.13 5.1320.281 20.139 20.032 19.764 19.959 20.235 A5.1S3 5.10 5.12 5.11 5.115.11 5.13 20.391 20.009 20.084 20.034 20.011 20.062 A5.1S4 5.13 5.155.16 5.17 5.16 5.15 20.713 20.390 20.383 20.233 20.546 20.562 A5.2S15.20 5.22 5.21 5.20 5.22 5.22 20.861 20.403 20.433 20.195 20.227 20.188A5.2S2 5.16 5.20 5.21 5.20 5.21 5.20 20.454 20.177 20.109 20.105 20.23420.010 A5.2S3 5.24 5.29 5.28 5.26 5.27 5.27 20.511 20.429 19.138 19.93518.444 20.125 A5.2S4 5.22 5.26 5.26 5.24 5.26 5.26 20.356 20.112 20.17519.822 20.107 19.963 A5.3S1 5.33 5.37 5.39 5.38 5.35 5.39 20.511 20.06820.057 19.996 20.253 20.045 A5.3S2 5.25 5.30 5.30 5.29 5.30 5.30 20.57620.357 20.191 20.175 20.378 20.179 A5.3S3 5.32 5.36 5.37 5.36 5.34 5.3720.609 20.276 20.178 20.182 20.107 20.101 A5.3S4 5.27 5.30 5.31 5.305.32 5.31 20.704 20.475 20.285 20.211 20.252 20.217 A5.4S1 5.40 5.425.44 5.41 5.41 5.44 20.418 20.068 19.956 19.930 19.908 19.826 A5.4S20.40 5.41 5.43 5.41 5.43 5.43 20.324 19.992 19.978 19.811 19.912 19.925A5.4S3 0.43 5.46 5.46 5.44 5.46 5.46 20.360 20.232 20.051 20.034 20.02019.966 A5.4S4 0.36 5.40 5.40 5.38 5.41 5.40 20.668 20.477 20.42

19.884 20.317 20.293 Actemra ® 6.46 6.49 6.51 5.49 5.48 6.31 19.76519.591 19.573 19.241 19.544 19.295

indicates data missing or illegible when filed3.2 SEC-HPLC Analysis after Thermal Stress Test

The acetate samples after thermal stress treatment are further analyzedusing SEC-HPLC. FIGS. 1, 2, and 3 show SEC-HPLC profile of TocilizumabIgG before being subjected to storage (To) or after being subjected tostorage at 4° C. or 25° C. for 6 months. The area of pre-peak, main-peakand post-peak were integrated to calculate the proportion of aggregates,monomers and fragments of Tocilizumab IgG, and the result of calculationwere presented in Tables 26 to 29.

Tables 26 to 29 demonstrate the percentage of Tocilizumab IgG monomers(main peak %), aggregates (HMW %), and fragments (LMW %) of the acetatesamples by the SEC-HPLC analysis, before being subjected to storage (T₀)or after being subjected to storage at 4° C., 25° C., or 40° C. for 3 to6 months.

As shown in Tables 26, 27-1, and 27-2, each of the acetate samples hassimilar percentage of Tocilizumab IgG monomers (main peak %), aggregates(HMW %), and fragments (LMW %). In Tables 26, 27-1, and 27-2, Actemra®has more aggregates (HMW %: 0.6˜0.8%) and aggregate increase (IncreasedHMW %: 0.11˜0.14%) than other samples (HMW %: 0.2˜0.35%; Increased HMW%: 0.01˜0.08%) either before being subjected to storage (To) or afterbeing subjected to storage at 4° C. for 3 to 6 months.

After storing at 25° C. for 3 months, the acetate samples show similarresults to the acetate samples before being subjected to storage (To)(see Table 28-1). However, as the storage time is prolonged to 6 months,the percentage of monomers (main peak %) slightly decreases, thepercentage of fragments (LMW %) slightly increases, and the shoulderappears (FIG. 3.). Moreover, the fragments and shoulder formation of theacetate samples decrease along with the increase in pH level, and thefragments and shoulder formation of the acetate samples increase alongwith the increase in sorbitol concentration (see Table 27-2). Thephenomena aforementioned could be observed more evidently when theacetate samples are subjected to storage at 40° C. for 3 months.

Table 29 also shows that Actemra® has more aggregates (HMW %: 1.59%) andhigher aggregate increase (Increased HMW %: 0.93%) than other acetatesamples (HMW %: 0.4-0.6%; Increased HMW %: 0.2-0.3%) after beingsubjected to 3 months of storage at 40° C. Further, the fragmentformation of the acetate samples decreased with the increase of pHlevel, and the acetate samples with higher sorbitol concentration showsmore monomer loss than the acetate samples with lower sorbitolconcentration at the same pH condition, after being stored at 40° C. for3 months.

TABLE 26 SEC-HPLC peak summary of the formulation candidates of Example2, before being subjected to storage (T₀) Sample Number HMW % Main peak% LMW % Sum area A5.1S1 0.23 99.75 0.02 34728150 A5.1S2 0.23 99.75 0.0237277772 A5.1S3 0.20 99.77 0.02 35029061 A5.1S4 0.23 99.75 0.02 38021887A5.2S1 0.22 99.75 0.02 35204088 A5.2S2 0.25 99.73 0.03 35213791 A5.2S30.25 99.73 0.02 35274716 A5.2S4 0.21 99.77 0.02 34901083 A5.3S1 0.2599.73 0.02 35001255 A5.3S2 0.25 99.72 0.02 36387499 A5.3S3 0.25 99.720.03 36456144 A5.3S4 0.25 99.72 0.02 35852816 A5.4S1 0.26 99.71 0.0235366994 A5.4S2 0.27 99.71 0.02 37254869 A5.4S3 0.26 99.71 0.02 35635718A5.4S4 0.26 99.71 0.02 37309692 Actemra ® 0.66 99.26 0.09 36579732

TABLE 27-1 SEC-HPLC peak summary of the formulation candidates ofExample 2, with a storage condition of 4° C. for 3 months SampleIncreased Main Increased Increased Number HMW % HMW % peak % Main-peak %LMW % LMW % Sum area A5.1S1 0.26 0.03 99.60 −0.15 0.14 0.12 36036.3A5.1S2 0.25 0.02 99.61 −0.14 0.14 0.12 36568.4 A5.1S3 0.24 0.04 99.60−0.17 0.16 0.14 36245.9 A5.1S4 0.25 0.02 99.57 −0.18 0.18 0.16 37061.9A5.2S1 0.26 0.04 99.60 −0.15 0.14 0.12 36841.7 A5.2S2 0.26 0.01 99.60−0.13 0.14 0.11 37926.5 A5.2S3 0.27 0.02 99.58 −0.15 0.15 0.13 36856A5.2S4 0.26 0.05 99.56 −0.21 0.18 0.16 36951.9 A5.3S1 0.29 0.04 99.57−0.16 0.14 0.12 36267.7 A5.3S2 0.28 0.03 99.58 −0.14 0.14 0.12 36895.7A5.3S3 0.29 0.04 99.58 −0.14 0.12 0.09 36833 A5.3S4 0.29 0.04 99.62−0.10 0.09 0.07 36381.8 A5.4S1 0.32 0.06 99.59 −0.12 0.10 0.08 36228.2A5.4S2 0.30 0.03 99.60 −0.11 0.10 0.08 36278.1 A5.4S3 0.30 0.04 99.58−0.13 0.11 0.09 36630.1 A5.4S4 0.29 0.03 99.58 −0.13 0.13 0.11 37237.9Actemra ® 0.80 0.14 99.06 −0.20 0.14 0.05 36661.2

TABLE 27-2 SEC-HPLC peak summary of the formulation candidates ofExample 2, with a storage condition of 4° C. for 6 months SampleIncrease Main Increased Increase Number HMW % HMW % peak % Main-peak %LMW % LMW % Sum area A5.1S1 0.26 0.03 99.62 −0.13 0.12 0.10 36700.1306A5.1S2 0.26 0.03 99.61 −0.14 0.13 0.11 36873.4974 A5.1S3 0.25 0.05 99.63−0.14 0.12 0.10 36997.9602 A5.1S4 0.27 0.04 99.61 −0.14 0.12 0.1037065.4542 A5.2S1 0.27 0.05 99.60 −0.15 0.13 0.11 38330.5265 A5.2S2 0.280.03 99.60 −0.13 0.13 0.10 41433.2793 A5.2S3 0.31 0.06 99.57 −0.16 0.120.10 34535.3855 A5.2S4 0.27 0.06 99.62 −0.15 0.12 0.10 36914.0760 A5.3S10.33 0.08 99.49 −0.24 0.17 0.15 37886.8534 A5.3S2 0.31 0.06 99.55 −0.170.14 0.12 38126.3745 A5.3S3 0.31 0.06 99.57 −0.15 0.13 0.10 37415.7769A5.3S4 0.29 0.04 99.57 −0.15 0.13 0.11 37675.9355 A5.4S1 0.32 0.06 99.53−0.18 0.14 0.12 36918.7516 A5.4S2 0.31 0.04 99.54 −0.17 0.14 0.1236891.8872 A5.4S3 0.31 0.05 99.54 −0.17 0.15 0.13 37506.6960 A5.4S4 0.300.04 99.50 −0.21 0.20 0.18 37805.3653 Actemra ® 0.77 0.11 98.99 −0.270.24 0.15 36135.7490

TABLE 28-1 SEC-HPLC peak summary of the formulation candidates ofExample 2, with a storage condition of 25° C. for 3 months SampleIncreased Main Increased Increased Number HMW % HMW % peak % Main-peak %LMW % LMW % Sum area A5.1S1 0.28 0.05 99.38 −0.37 0.35 0.33 35789.3A5.1S2 0.27 0.04 99.38 −0.37 0.35 0.33 36407.1 A5.1S3 0.25 0.05 99.37−0.4 0.38 0.36 36364.4 A5.1S4 0.27 0.04 99.32 −0.43 0.41 0.39 36821.6A5.2S1 0.29 0.07 99.36 −0.39 0.35 0.33 36982.7 A5.2S2 0.29 0.04 99.36−0.37 0.35 0.32 36348.3 A5.2S3 0.32 0.07 99.34 −0.39 0.33 0.31 36022.5A5.2S4 0.28 0.07 99.33 −0.44 0.39 0.37 36027.7 A5.3S1 0.33 0.08 99.35−0.38 0.32 0.3 36072.8 A5.3S2 0.31 0.06 99.36 −0.36 0.33 0.31 36950.7A5.3S3 0.32 0.07 99.34 −0.38 0.34 0.31 36617.7 A5.3S4 0.31 0.06 99.34−0.38 0.35 0.33 36504 A5.4S1 0.35 0.09 99.34 −0.37 0.31 0.29 36125.8A5.4S2 0.34 0.07 99.36 −0.35 0.31 0.29 36430.5 A5.4S3 0.34 0.08 99.33−0.38 0.33 0.31 36532.8 A5.4S4 0.32 0.06 98.32 −0.39 0.36 0.34 36961.9Actemra ® 0.83 0.17 98.82 −0.44 0.35 0.26 35899.8

TABLE 28-2 SEC-HPLC peak summary of the formulation candidates ofExample 2, with a storage condition of 25° C. for 6 months SampleIncreased Increased Main- Increased Increased Number HMW % HMW % Mainpeak % peak % Shoulder % Shoulder % LMW % LMW % Sum area A5.1S1 0.300.07 97.06 −2.69 2.07 2.07 0.57 0.55 39150.1953 A5.1S2 0.30 0.07 97.04−2.71 2.09 2.09 0.58 0.56 39098.1052 A5.1S3 0.28 0.08 97.04 −2.73 2.112.11 0.57 0.55 37678.7645 A5.1S4 0.30 0.07 96.96 −2.79 2.16 2.16 0.570.55 37576.8264 A5.2S1 0.32 0.10 97.14 −2.61 2.00 2.00 0.54 0.5237759.4751 A5.2S2 0.31 0.06 97.12 −2.61 2.02 2.02 0.55 0.52 37465.7636A5.2S3 0.32 0.07 97.12 −2.61 2.02 2.02 0.55 0.53 37986.3794 A5.2S4 0.310.10 97.01 −2.76 2.13 2.13 0.55 0.53 36921.7585 A5.3S1 0.36 0.11 97.27−2.46 1.87 1.87 0.50 0.48 37376.7372 A5.3S2 0.35 0.10 97.23 −2.49 1.911.91 0.52 0.50 41207.2882 A5.3S3 0.36 0.11 97.25 −2.47 1.87 1.87 0.510.48 37445.9972 A5.3S4 0.35 0.10 97.02 −2.70 2.08 2.08 0.54 0.5237688.2849 A5.4S1 0.39 0.13 97.36 −2.35 1.78 1.78 0.48 0.46 37215.9016A5.4S2 0.38 0.11 97.34 −2.37 1.80 1.80 0.49 0.47 36885.3817 A5.4S3 0.380.12 97.36 −2.35 1.78 1.78 0.48 0.46 37146.1753 A5.4S4 0.36 0.10 97.33−2.38 1.82 1.82 0.49 0.47 37572.7428 Actemra ® 0.89 0.23 96.79 −2.471.79 1.79 0.54 0.45 35998.7191

TABLE 29 SEC-HPLC peak summary of the formulation candidates of Example2, with a storage condition of 40° C. for 3 months Increased SampleIncreased Main Main- Increased Number HMW % HMW % peak % peak % Shoulder% LMW % LMW % Sum area A5.1S1 0.52 0.29 93.1 −6.65 4.7 1.69 1.6735252.1818 A5.1S2 0.45 0.22 93.15 −6.6 4.73 1.67 1.65 36033.6966 A5.1S30.41 0.21 93.02 −6.75 4.96 1.61 1.59 35958.8232 A5.1S4 0.58 0.35 92.55−7.2 5.25 1.62 1.6 36766.0109 A5.2S1 0.47 0.25 93.49 −6.26 4.47 1.571.55 36405.2105 A5.2S2 0.46 0.21 93.47 −6.26 4.49 1.58 1.55 36634.0103A5.2S3 0.46 0.21 93.47 −6.26 4.6 1.47 1.45 36099.1330 A5.2S4 0.43 0.2293.1 −6.67 4.95 1.52 1.5 35831.3562 A5.3S1 0.52 0.27 93.91 −5.82 4.151.43 1.41 36656.3546 A5.3S2 0.5 0.25 93.72 −6 4.29 1.49 1.47 36394.4932A5.3S3 0.54 0.29 93.1 −6.62 4.83 1.54 1.51 36389.1406 A5.3S4 0.51 0.2692.77 −6.95 5.13 1.59 1.57 36247.9774 A5.4S1 0.6 0.34 93.71 −6 4.22 1.461.44 35977.0024 A5.4S2 0.51 0.24 94.31 −5.4 3.87 1.31 1.29 35336.2693A5.4S3 0.52 0.26 93.84 −5.87 4.33 1.32 1.3 36072.3756 A5.4S4 0.52 0.2693.68 −6.03 4.47 1.33 1.31 35562.8917 Actemra ® 1.59 0.93 93.26 −6 3.821.34 1.25 35040.7368

The above test data obtained from Example 2 is demonstrated that theacetate samples have less protein aggregates formation than Actemra®,after a long-term storage at 4° C., 25° C., or 40° C. The acetatesamples with higher pH level has less shoulder and fragments formation,and the acetate samples containing higher sorbitol concentration hashigher monomer loss and higher shoulder and fragments formation, afterthe long-term storage at 25° C. and 40° C.

As evidenced by the above results, the acetate buffer having higher pHlevel and lower sorbitol concentration improve the thermostability inthe Tocilizumab IgG formulation. Therefore, the formulation candidatesof the present disclosure having higher pH level and lower sorbitolconcentration are better formulation than Actemra®.

In sum, according to various embodiments of the present disclosure, theantibody-containing aqueous formulations having pH values of 4.5 to 6.5can stabilize and reduce aggregate formation of the anti-interleukin-6receptor antibody in the antibody-containing aqueous formulation. Theantibody-containing aqueous formulations provided by the presentdisclosure are thermostable, therefore can be stored at 4° C., 25° C.,or 40° C. for at least 3 months.

Previous descriptions are only embodiments of the present disclosure andare not intended to limit the scope of the present disclosure. Manyvariations and modifications according to the claims and specificationof the disclosure are still within the scope of the claimed disclosure.In addition, each of the embodiments and claims does not have to achieveall the advantages or characteristics disclosed. Moreover, the abstractand the title only serve to facilitate searching patent documents andare not intended in any way to limit the scope of the claimeddisclosure.

What is claimed is:
 1. An antibody-containing aqueous formulation,comprising: a therapeutically effective amount of an anti-interleukin-6receptor antibody; a protein stabilizer; a surfactant; and a buffer,wherein the buffer is an acetate buffer or a histidine buffer, and theantibody-containing aqueous formulation falling within a pH range of 4.5to 6.5.
 2. The antibody-containing aqueous formulation according toclaim 1, wherein a concentration of the Tocilizumab IgG falls within arange of 5 mg/ml to 30 mg/ml.
 3. The antibody-containing aqueousformulation according to claim 1, wherein the protein stabilizer issucrose or trehalose, and a concentration of the protein stabilizerfalls within a range of 1% (w/v) to 10% (w/v).
 4. Theantibody-containing aqueous formulation according to claim 1, whereinthe surfactant is polysorbate 80, polysorbate 20 or poloxamer, and aconcentration of the surfactant falls within a range of 0.01% (w/v) to0.05% (w/v).
 5. The antibody-containing aqueous formulation according toclaim 1, wherein a concentration of the buffer falls within a range of10 mM to 30 mM.
 6. The antibody-containing aqueous formulation accordingto claim 1, wherein the buffer is an acetate buffer, and a pH of theantibody-containing aqueous formulation falls within a range of 5.0 to6.0.
 7. The antibody-containing aqueous formulation according to claim1, wherein the buffer is a histidine buffer, and a pH of theantibody-containing aqueous formulation falls within a range of 5.5 to6.5.
 8. The antibody-containing aqueous formulation according to claim1, further comprising a tonicifier.
 9. The antibody-containing aqueousformulation according to claim 8, wherein the tonicifier is 1% (w/v) to4% (w/v) of sorbitol.
 10. The antibody-containing aqueous formulationaccording to claim 1, wherein the anti-interleukin-6 receptor antibodyis 5 mg/ml to 30 mg/ml of Tocilizumab IgG, the protein stabilizer is 4%(w/v) to 7% (w/v) of sucrose, the surfactant is 0.01% (w/v) to 0.05%(w/v) of polysorbate 80, the buffer is 10 mM to 30 mM of acetate buffer,and the antibody-containing aqueous formulation has a pH ranging between5.0 and 6.0.
 11. The antibody-containing aqueous formulation accordingto claim 1, wherein the anti-interleukin-6 receptor antibody is 5 mg/mlto 30 mg/ml of Tocilizumab IgG, the protein stabilizer is 4% (w/v) to 7%(w/v) of sucrose, the surfactant is 0.01% (w/v) to 0.05% (w/v) ofpolysorbate 80, the buffer is 10 mM to 30 mM of histidine buffer, andthe antibody-containing aqueous formulation has a pH ranging between 5.5and 6.5.
 12. The antibody-containing aqueous formulation according toclaim 1, wherein the anti-interleukin-6 receptor antibody is 20 mg/ml ofTocilizumab IgG, the protein stabilizer is 7% (w/v) of sucrose, thesurfactant is 0.03% (w/v) of polysorbate 80, the buffer is 20 mM ofhistidine buffer, and the antibody-containing aqueous formulation has apH ranging between 5.5 and 6.5.
 13. The antibody-containing aqueousformulation according to claim 1, further comprising a tonicifier, andthe tonicifier is 1% (w/v) to 4% (w/v) of sorbitol.
 14. Theantibody-containing aqueous formulation according to claim 1, whereinthe antibody-containing aqueous formulation forms less than 5% ofaggregates of the anti-interleukin-6 receptor antibody after 3 months ofstorage at 40° C.
 15. The antibody-containing aqueous formulationaccording to claim 1, wherein at least 90% of the anti-interleukin-6receptor antibody in the antibody-containing aqueous formulation is inmonomer form after 3 months of storage at 40° C.
 16. Theantibody-containing aqueous formulation according to claim 1, whereinthe antibody-containing aqueous formulation forms less than 5% ofaggregates and at least 90% of the anti-interleukin-6 receptor antibodyin the antibody-containing aqueous formulation is in monomer form after3 months of storage at 40° C.
 17. The antibody-containing aqueousformulation according to claim 1, wherein the antibody-containingaqueous formulation is administered to a subject by intravenousinjection or infusion.
 18. A method of treating aninterleukin-6-mediated disease, comprising administering theantibody-containing aqueous formulation of claim 1 to a mammalian body.19. The method according to claim 18, wherein the interleukin-6-mediateddisease is rheumatoid arthritis, systemic juvenile idiopathic arthritis,polyarticular juvenile idiopathic arthritis, Still's disease,Castleman's disease, giant cell arteritis, system sclerosis, cytokinerelease syndrome, polymyositis, dermatomyositis, familial mediterraneanfever, polymyalgia rheumatic, Schnitzler syndrome, pulmonary arterialhypertension, pain, or B-cell chronic lymphocytic leukemia.
 20. A methodof suppressing aggregate formation of anti-interleukin-6 receptorantibody in an antibody-containing aqueous formulation, comprising stepsof: formulating the anti-Interleukin-6 receptor antibody with an aqueoussolution having a pH ranging between 4.5 and 6.5, wherein the aqueoussolution comprises a buffer, a stabilizer, and a surfactant, and thebuffer is an acetate buffer or a histidine buffer.